Uromodulin is a zona pellucida-type protein essentially produced in the thick ascending limb (TAL) of the mammalian kidney. It is the most abundant protein in normal urine. Defective uromodulin ...processing is associated with various kidney disorders. The luminal release and subsequent polymerization of uromodulin depend on its cleavage mediated by the serine protease hepsin. The biological relevance of a proper cleavage of uromodulin remains unknown. Here we combined in vivo testing on hepsin-deficient mice, ex vivo analyses on isolated tubules and in vitro studies on TAL cells to demonstrate that hepsin influence on uromodulin processing is an important modulator of salt transport via the sodium cotransporter NKCC2 in the TAL. At baseline, hepsin-deficient mice accumulate uromodulin, along with hyperactivated NKCC2, resulting in a positive sodium balance and a better adaptation to water deprivation. In conditions of high salt intake, defective uromodulin processing predisposes hepsin-deficient mice to a salt-wasting phenotype, with a decreased salt sensitivity. These modifications are associated with intracellular accumulation of uromodulin, endoplasmic reticulum-stress and signs of tubular damage. These studies expand the physiological role of hepsin and uromodulin and highlight the importance of hepsin-mediated processing of uromodulin for kidney tubule homeostasis and salt sensitivity.
Missense mutations in the uromodulin (UMOD) gene cause autosomal dominant tubulointerstitial kidney disease (ADTKD), one of the most common monogenic kidney diseases. The unknown impact of the ...allelic and gene dosage effects and fate of mutant uromodulin leaves open the gap between postulated gain‐of‐function mutations, end‐organ damage and disease progression in ADTKD. Based on two prevalent missense UMOD mutations with divergent disease progression, we generated UmodC171Y and UmodR186S knock‐in mice that showed strong allelic and gene dosage effects on uromodulin aggregates and activation of ER stress and unfolded protein and immune responses, leading to variable kidney damage. Deletion of the wild‐type Umod allele in heterozygous UmodR186S mice increased the formation of uromodulin aggregates and ER stress. Studies in kidney tubular cells confirmed differences in uromodulin aggregates, with activation of mutation‐specific quality control and clearance mechanisms. Enhancement of autophagy by starvation and mTORC1 inhibition decreased uromodulin aggregates. These studies substantiate the role of toxic aggregates as driving progression of ADTKD‐UMOD, relevant for therapeutic strategies to improve clearance of mutant uromodulin.
Synopsis
Representative missense mutations in the UMOD gene causing ADTKD differentially drive the formation of mutant uromodulin aggregates, impacting on kidney damage and disease progression. Enhancement of autophagy decreased uromodulin aggregates, relevant for therapeutic strategies in ADTKD.
Two Umod KI models show strong allelic and gene dosage effects on uromodulin aggregates and activation of inflammation and fibrosis, leading to variable kidney damage.
The wild‐type Umod allele protects against the formation of uromodulin aggregates.
Studies in kidney tubular cells support mutation‐specific effects on uromodulin aggregates and activation of quality control and clearance mechanisms.
Enhancement of autophagy by mTORC1 inhibition decreased uromodulin aggregates.
Representative missense mutations in the UMOD gene causing ADTKD differentially drive the formation of mutant uromodulin aggregates, impacting on kidney damage and disease progression. Enhancement of autophagy decreased uromodulin aggregates, relevant for therapeutic strategies in ADTKD.
Current understanding of fibrosis remains incomplete despite the increasing burden of related diseases. Preclinical models are used to dissect the pathogenesis and dynamics of fibrosis, and to ...evaluate anti-fibrotic therapies. These studies require objective and accurate measurements of fibrosis. Existing histological quantification methods are operator-dependent, organ-specific, and/or need advanced equipment. Therefore, we developed a robust, minimally operator-dependent, and tissue-transposable digital method for fibrosis quantification. The proposed method involves a novel algorithm for more specific and more sensitive detection of collagen fibers stained by picrosirius red (PSR), a computer-assisted segmentation of histological structures, and a new automated morphological classification of fibers according to their compactness. The new algorithm proved more accurate than classical filtering using principal color component (red-green-blue; RGB) for PSR detection. We applied this new method on established mouse models of liver, lung, and kidney fibrosis and demonstrated its validity by evidencing topological collagen accumulation in relevant histological compartments. Our data also showed an overall accumulation of compact fibers concomitant with worsening fibrosis and evidenced topological changes in fiber compactness proper to each model. In conclusion, we describe here a robust digital method for fibrosis analysis allowing accurate quantification, pattern recognition, and multi-organ comparisons useful to understand fibrosis dynamics.
Integrins are heterodimers anchoring cells to the surrounding extracellular matrix (ECM), an active and complex process mediating a series of inside-out and outside-in stimuli regulating cellular ...turn-over, tissue growth and architecture. Itgb1 is the main subunit of the renal integrins and it is critical for renal development. This study aims to investigate the role of Itgb1 in the adult renal epithelial cells by knocking down Itgb1 in PAX8 expressing cells.
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cKO mice develop a progressively worsening proteinuria and renal abnormalities leading to severe renal failure and hypertension. This phenotype is also associated with severe dysfunction of distal nephron and polyuria. To further investigate whether distal nephron involvement was primarily related to Itgb1 suppression or secondary to renal failure, an
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cKO mouse model was generated. These mice lack Itgb1 expression in AQP2 expressing cells. They do not show any developmental alteration, but 1 month old mice are resistant to dDAVP administration and finally, at 2 months of age, they develop overt polyuria. This phenotype is due to primary collecting duct (CD) cells
. The entire architecture of the outer medulla is altered, with loss of the typical organization pattern of vascular and tubular bundles alternation. Indeed, even though not primarily affected by genetic ablation, the TAL is secondarily affected in this model. It is sufficient to suppress Itgb1 expression in the CD in order to stimulate proliferation and then disappearance of neighboring TAL cells. This study shows that cell to cell interaction through the ECM is critical for architecture and function maintenance of the outer medulla and that Itgb1 is crucial for this process.
Uromodulin, the most abundant protein in normal urine, is produced by cells lining the thick ascending limb (TAL) of the loop of Henle. Uromodulin regulates the activity of the potassium channel ROMK ...in TAL cells. Common variants in KCNJ1, the gene encoding ROMK, are associated with urinary levels of uromodulin in population studies. Here, we investigated the functional link between ROMK and uromodulin in Kcnj1 knock-out mouse models, in primary cultures of mouse TAL (mTAL) cells, and in patients with Bartter syndrome due to KCNJ1 mutations. Both global and kidney-specific Kcnj1 knock-out mice showed reduced urinary levels of uromodulin paralleled by increased levels in the kidney, compared to wild-type controls. Pharmacological inhibition and genetic deletion of ROMK in mTAL cells caused a reduction in apical uromodulin excretion, reflected by cellular accumulation. In contrast, NKCC2 inhibition showed no effect on uromodulin processing. Patients with Bartter syndrome type 2 showed reduced urinary uromodulin levels compared to age and gender matched controls. These results demonstrate that ROMK directly regulates processing and release of uromodulin by TAL cells, independently from NKCC2. They support the functional link between transport activity and uromodulin in the TAL, relevant for blood pressure control and urinary concentrating ability.
Heterozygous mutations in UMOD encoding the urinary protein uromodulin are the most common genetic cause of autosomal dominant tubulointerstitial kidney disease (ADTKD). We describe the exceptional ...case of a patient from a consanguineous family carrying a novel homozygous UMOD mutation (p.C120Y) affecting a conserved cysteine residue within the EGF-like domain III of uromodulin. Comparison of heterozygote and homozygote mutation carriers revealed a gene dosage effect with unprecedented low levels of uromodulin and aberrant uromodulin fragments in the urine of the homozygote proband. Despite an amplified biological effect of the homozygote mutation, the proband did not show a strikingly more severe clinical evolution nor was the near absence of urinary uromodulin associated with urinary tract infections or kidney stones.
Uromodulin, the most abundant protein excreted in normal urine, plays major roles in kidney physiology and disease. The mechanisms regulating the urinary excretion of uromodulin remain essentially ...unknown.
We conducted a meta-analysis of genome-wide association studies for raw (uUMOD) and indexed to creatinine (uUCR) urinary levels of uromodulin in 29,315 individuals of European ancestry from 13 cohorts. We tested the distribution of candidate genes in kidney segments and investigated the effects of keratin-40 (KRT40) on uromodulin processing.
Two genome-wide significant signals were identified for uUMOD: a novel locus (
1.24E-08) over the
gene coding for KRT40, a type 1 keratin expressed in the kidney, and the
locus (
2.17E-88), with two independent sets of single nucleotide polymorphisms spread over
and
. Two genome-wide significant signals for uUCR were identified at the
locus and at the novel
locus previously associated with kidney function. The effect sizes for rs8067385, the index single nucleotide polymorphism in the
locus, were similar for both uUMOD and uUCR. KRT40 colocalized with uromodulin and modulating its expression in thick ascending limb (TAL) cells affected uromodulin processing and excretion.
Common variants in
,
,
, and
associate with the levels of uromodulin in urine. The expression of KRT40 affects uromodulin processing in TAL cells. These results, although limited by lack of replication, provide insights into the biology of uromodulin, the role of keratins in the kidney, and the influence of the
locus on kidney function.
The kidney-specific gene
encodes for uromodulin, the most abundant protein excreted in normal urine. Rare large-effect variants in
cause autosomal dominant tubulointerstitial kidney disease (ADTKD), ...while common low-impact variants strongly associate with kidney function and the risk of chronic kidney disease (CKD) in the general population. It is unknown whether intermediate-effect variants in
contribute to CKD. Here, candidate intermediate-effect
variants were identified using large-population and ADTKD cohorts. Biological and phenotypical effects were investigated using cell models, in silico simulations, patient samples, and international databases and biobanks. Eight
missense variants reported in ADTKD are present in the Genome Aggregation Database (gnomAD), with minor allele frequency (MAF) ranging from 10
to 10
. Among them, the missense variant p.Thr62Pro is detected in ∼1/1,000 individuals of European ancestry, shows incomplete penetrance but a high genetic load in familial clusters of CKD, and is associated with kidney failure in the 100,000 Genomes Project (odds ratio OR = 3.99 1.84 to 8.98) and the UK Biobank (OR = 4.12 1.32 to 12.85). Compared with canonical ADTKD mutations, the p.Thr62Pro carriers displayed reduced disease severity, with slower progression of CKD and an intermediate reduction of urinary uromodulin levels, in line with an intermediate trafficking defect in vitro and modest induction of endoplasmic reticulum (ER) stress. Identification of an intermediate-effect
variant completes the spectrum of
-associated kidney diseases and provides insights into the mechanisms of ADTKD and the genetic architecture of CKD.
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